ADP-glucose pyrophoshorylase (ADP-Glc PPase) catalyzes the first and limiting step in starch biosynthesis. In plants, the enzyme is composed of two types of subunits (small and large) and is allosterically regulated by 3-phosphoglycerate and phosphate. The pattern of expression and sugar regulation of the six Arabidopsis thaliana ADP-Glc PPase-encoding genes (two small subunits, ApS1 and ApS2; and four large subunits, ApL1-ApL4) has been studied. Based on mRNA expression, ApS1 is the main small subunit or catalytic isoform responsible for ADP-Glc PPase activity in all tissues of the plant. Large subunits play a regulatory role, and the data presented define a clear functional distinction among them. ApL1 is the main large subunit in source tissues, whereas ApL3 and, to a lesser extent, ApL4 are the main isoforms present in sink tissues. Thus, in source tissues, ADP-Glc PPase would be finely regulated by the 3-phosphoglycerate/phosphate ratio, whereas in sink tissues, the enzyme would be dependent on the availability of substrates for starch synthesis. Sugar regulation of ADP-Glc PPase genes is restricted to ApL3 and ApL4 in leaves. Sugar induction of ApL3 and ApL4 transcription in leaves allows the establishment of heterotetramers less sensitive to the allosteric effectors, resembling the situation in sink tissues. The results presented on the expression pattern and sugar regulation allow us to propose a gene evolution model for the Arabidopsis ADP-Glc PPase gene family.

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO2-enriched atmosphere [0.7% (v/v) CO2] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO2 to air. F1 plants of back crosses to wild type were viable in air and F2 populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO2 conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO2 conditions.

Granule-bound starch synthase (GBSSI) is one of the most extensively studied enzymes of the starch synthesis pathway and its role in the synthesis of amylose has been well established. However, few studies have been carried out to characterize the regulation of GBSSI gene. Regulation of starch synthesis genes is especially interesting in photosynthetic tissues, where starch is subjected to a periodical alternation of synthesis and degradation during the day/night cycle. In this report we show a circadian oscillation of GBSSI mRNA levels in leaves of Arabidopsis during the day/night cycle, and provide evidence that GBSSI expression is controlled by the transcription factors CCA1 and LHY. Over-expression of both CCA1 and LHY genes causes the elimination of GBSSI mRNA oscillation. Binding shift assays indicate that this control may be exerted through a direct interaction of those regulatory proteins with the GBSSI promoter. Oscillation is not observed on the GBSSI protein levels, which remains constant along the cycle. However, GBSSI activity shows a clear oscillation with a period of 24 h that is altered in transgenic plants over-expressing CCA1. Possible mechanisms controlling GBSSI activity during the day/night cycle are discussed.